Endothelial cell damage and dysfunction are early events in the pathogenesis of atherosclerosis. Although they both have been observed in patients with diabetes, the mechanisms responsible for their occurrence is unclear. This project examines the hypothesis that an imbalance between intracellular fatty acid availability and oxidation induced by hyperglycemia, elevated plasma free fatty acids (FFA) and the two in combination is a key pathogenetic event. In support of this notion, we have shown that hyperglycemia (30mM glucose)-induced apoptosis in human umbilical vein endothelial cells (HUVEC) is preceded by inhibition of fatty acid oxidation, an increase in the synthesis of diacylglycerol, and an impaired ability of insulin to activate Akt. We have also found that all of these changes are prevented by adding to the incubation medium, AICAR, an activator of AMP-activated protein kinase. Furthermore, incubation with the fatty acid palmitate (0.5mM) in a normoglycemic medium also caused apoptosis, and this too was prevented by AICAR. To assess our basic hypothesis, studies will be carried out in HUVEC and/or human aortic endothelial cells with the following aims: (1) To characterize glucose and fatty acid metabolism in endothelium and determine how they are altered when AMPK is activated by metformin, AICAR or infection with a constitutively active AMPK linked to adenovirus; (2) To determine whether sustained hyperglycemia causes endothelial cell damage (apoptosis) and impairs insulin signaling by altering DAG-PKC signaling, ceramide synthesis and/or oxidative stress; (3) To prove that AMPK activation is responsible for the protective effect of AICAR and to determine how it works; and (4) To evaluate whether excess FFA (palmitate) causes cell damage by a similar mechanism and if its effects are additive to those of hyperglycemia. These studies will provide novel information as to how the metabolic stresses associated with diabetes cause damage to the endothelium. They should also yield insights into how the endothelial cell attempts to protect itself against these stresses and whether AMPK is a potential target for therapy.